Targeted therapy for head and neck cancer: signaling pathways and clinical studies

Head and neck cancer (HNC) is malignant, genetically complex and difficult to treat and is the sixth most frequent cancer, with tobacco, alcohol and human papillomavirus being major risk factors. Based on epigenetic data, HNC is remarkably heterogeneous, and treatment remains challenging. There is a lack of significant improvement in survival and quality of life in patients with HNC. Over half of HNC patients experience locoregional recurrence or distal metastasis despite the current multiple traditional therapeutic strategies and immunotherapy. In addition, resistance to chemotherapy, radiotherapy and some targeted therapies is common. Therefore, it is urgent to explore more effective and tolerable targeted therapies to improve the clinical outcomes of HNC patients. Recent targeted therapy studies have focused on identifying promising biomarkers and developing more effective targeted therapies. A well understanding of the pathogenesis of HNC contributes to learning more about its inner association, which provides novel insight into the development of small molecule inhibitors. In this review, we summarized the vital signaling pathways and discussed the current potential therapeutic targets against critical molecules in HNC, as well as presenting preclinical animal models and ongoing or completed clinical studies about targeted therapy, which may contribute to a more favorable prognosis of HNC. Targeted therapy in combination with other therapies and its limitations were also discussed.


INTRODUCTION
Head and neck cancer (HNC) is the sixth most frequent cancer type worldwide, with over 870,000 new cases and 440,000 deaths in 2020. 1 Head and neck squamous cell carcinoma (HNSCC) is the most common type of HNC, accounting for approximately 90% of HNC cases, and primarily originates from the mucosal epithelium of the oral cavity, pharynx and larynx. 2 The incidence of HNSCC is increasing and is predicted to rise to 1.08 million new cases per year by 2030. 3 Exposure to tobacco-derived carcinogens and chronic heavy alcohol consumption are high-risk factors for HNC globally. Recently, increasing tumors in the oropharynx have been associated with previous infection with human papillomavirus (HPV, mainly HPV-16 and HPV-18), especially in Western countries. 4 As the most common oncogenic HPVs, HPV-16 and HPV-18 infection can be prevented by commercialized HPV vaccines. Therefore, it is feasible to prevent HPV-positive HNSCC by mass vaccination worldwide, just as it is to prevent cervical cancer. Cancers that arise in the oral cavity and larynx are mostly related to smoking and are classified as HPV-negative HNSCC. 5 Epstein-Barr virus infection is another risk factor that can contribute to the carcinogenesis of nasopharyngeal carcinoma (NPC). 6 There are no effective screening strategies for HNC, and most patients are often diagnosed at late stages. HNC is remarkably heterogeneous, and its treatment remains a challenge. Treatment of HNC patients requires aggressive multimodality approaches, including surgery followed by radiotherapy alone or with chemotherapy (known as chemoradiotherapy or chemoradiation) for oral cavity cancers and primary chemoradiotherapy for pharynx and larynx cancers. HPV-positive HNSCC usually displays a more favorable clinical outcome than HPV-negative HNSCC, resulting in the adaptation in the eighth edition of the tumor-node-metastasis (TNM) staging to include p16INK4A immunostaining to indicate HPV status. 2 Recently, two immune checkpoint inhibitors, pembrolizumab and nivolumab, have been approved by the Food and Drug Administration (FDA) for the treatment of recurrent or metastatic HNSCC (R/M-HNSCC), and pembrolizumab is a first-line therapy for unresectable tumors. [7][8][9] However, the prognosis remains poor. There is a lack of significant improvement in survival, and over half of HNSCC patients experience locoregional recurrence or distal metastasis. 5,10 In addition, patients receiving chemoradiotherapy may exhibit serious side effects and a poor quality of life. [11][12][13][14] Therefore, there is an urgent need to explore more effective and tolerable strategies to improve the clinical outcomes of HNC patients.
In recent decades, great success has been achieved in targeted therapy of HNC, which can accurately identify and kill cancer cells with low toxicity and side effects (Fig. 1). In 1984, Hendler et al. discovered a 2.5-to 5-fold increase in the expression of epidermal growth factor receptor (EGFR, HER1 or ErbB1) in human squamous cell lung cancers and epidermoid head and neck cancers. 15 EGFR belongs to the HER/ErbB family (consisting of HER/EGFR/ErbB 1 to 4) of receptor tyrosine kinases (RTKs), the activation of which leads to proliferation and metastasis of malignant cells and increased angiogenesis. [16][17][18][19] Early in 2001, the efficacy of cetuximab was investigated in squamous cell carcinomas in vivo, which also enhanced the efficacy of radiotherapy. [20][21][22] Shortly thereafter, cetuximab showed dose-dependent pharmacokinetics, tolerability, and biologic activity when combined with cisplatin in patients with advanced tumors overexpressing EGFR. 23 Further studies indicated that cetuximab is an effective radiation sensitizer, 24,25 resulting in the FDA approval of cetuximab plus radiotherapy for the treatment of locally advanced HNSCC (LA-HNSCC) patients in 2006. The addition of cetuximab to platinum-based chemotherapy increased the median overall survival (OS) to 10.1 months, which makes the EXTREME protocol (cetuximab, cisplatin, or carboplatin, 5-fluorouracil) the standard first-line treatment for R/M-HNSCC patients. 26 Although cetuximab increased the clinical efficacy and safety of conventional chemotherapy/radiotherapy, there is still much room for improvement in clinical outcomes and quality of life. 27 Current efforts are focusing on developing more potent and safe agents targeting EGFR and other signaling pathways, including vascular endothelial growth factor receptor (VEGFR), signaling, phosphatidylinositol 3-kinase (PI3K) signaling, and hepatocyte growth factor receptor (c-MET) signaling pathways.
In this review, we summarized the vital signaling pathways and discussed the current potential therapeutic targets in HNSCC as well as presenting preclinical animal models and ongoing or completed clinical studies about targeted therapy, which may contribute to a more favorable prognosis of HNSCC.

THE GENETIC LANDSCAPE IN HEAD AND NECK CANCER
EGFR pathway EGFR is a transmembrane glycoprotein and a cell surface receptor and is the primary member of the HER/ErbB family responding to RTKs. In HNC, EGFR mutation and amplification are rare. However, EGFR is overexpressed in~80% of HNSCC cases and is closely related to poor prognosis. 28 EGFR binds with HER family ligands involving epidermal growth factor (EGF), heparin binding-EGF, amphiregulin, transforming growth factor-alpha (TGF-α), epiregulin, and betacellulin, leading to a signal transduction cascade thereby promoting tumor proliferation, invasion, angiogenesis and metastasis and determining the outcomes of diseases. 29 Approaches to activate EGFR are multitudinous in head and neck cancer. The autocrine or paracrine effects of EGFR ligands, increasing the production of amphiregulin and TGF-α in response to tobacco smoke, activating G-protein-coupled receptors (GPCRs) and increasing the GPCR ligand PGE2 are involved in EGFR activation. 30,31 The EGFR pathway is complicated and multidimensional. The binding between EGFR and its ligand leads to homodimerization or heterodimerization with HER2, HER3, or other RTKs, such as insulin-like growth factor (IGF)-1R or MET. 32 The downstream signal transduction cascades, including the JAK/ STAT, PI3K/AKT, MAPK, PLCγ/PKC, and Src pathways, and the crosstalk among these signals are potential and attractive targets for HNC. 30 In addition to the membrane-bound form, EGFR is supposed to translocate to the cell nucleus and play multiple roles. EGFR can return to the cell membrane surface and undergo signal transduction and function. Nuclear EGFR is also associated with lysosome degradation, leading to downstream signal activation. 33 Moreover, nuclear EGFR can serve as a transcription factor, binding to several gene promotors (cyclo-oxygenase 2 (COX2), inducible nitric oxide synthase (iNOS) and cyclin D1) and DNAbinding transcription cofactors (signal transducer and activator of transcription (STAT3/5) and E2F1), along with PCNA and DNA-PK phosphorylation. 34 EGFR heterointeraction with Axl has been demonstrated to enhance oncogenic and invasive potential. 35 The Fig. 1 Timeline of treatment regimens and targeted therapy development in head and neck cancer and further investigation. Early on, surgery was first used to treat head and neck cancer. With further investigation, more therapies have been used to treat head and neck cancer. In the 1980s, an advanced understanding of HNSCC was made, and some major discoveries were made between the 1980s and 2020s. The prognosis of head and neck cancers was slightly improved but unsatisfactory. Some discoveries might first be made in cancers other than head and neck cancers, and the same discoveries were identified in head and neck cancers after years. More agents were found and approved in head and neck cancers, which may dramatically improve the prognosis of head and neck cancer patients. DNA deoxyribonucleic acid, EGFR epidermal growth factor receptor, HPV human papillomavirus, HNSCC head and neck squamous cell carcinoma, FDA Food and Drug Administration, IMRT intensity modulated radiation therapy Fig. 2 The EGFR signaling pathway, PI3K/AKT/mTOR pathway, MAPK pathway, STAT pathway, and MET pathway in head and neck cancer. EGFR epidermal growth factor receptor, TGF-α transforming growth factor-alpha, EGF epidermal growth factor, RTKs receptor tyrosine kinases, HER2/3 human epidermal growth factor receptor 2/3, c-MET c-mesenchymal-epithelial transition factor, IGF-1R insulin-like growth factor 1 receptor, PGE2 prostaglandin E2, GPCR G-protein-coupled receptor, PLCγ phospholipase c-γ, PKC protein kinase C, JAK Jenus-activated kinase; STAT3/5, signal transducer and activator of transcription 3/5, PTEN phosphatase and tensin homolog, PI3K phosphoinositide 3-kinase, PIP2 phosphatidylinositol 4,5-bisphosphate, PIP3 phosphatidylinositol 3,4,5-trisphosphate, AKT serine/threonine-specific protein kinase, mTOR mammalian target of rapamycin, DNMT-1 DNA methyltransferase 1, SOS son of sevenless, GRB2 growth factor receptor-bound protein 2, SHC SRC homology domain c-terminal adaptor homolog, ERK: extracellular signal-regulated kinase, MAP2K6 mitogen-activated protein kinase-kinase 6, PAK1 p21-activated kinase 1, DUSP dual-specificity phosphatases, PCNA proliferation cell nuclear antigen, DNA-PK DNAdependent protein kinase 2/3/4), and dual-specificity phosphatases (DUSP3/5/6/7/9), which are specific negative regulators of ERK. 70,71 Activation of several kinases, including BRAF, KRAS, HRAS, and ERK1/2, has been demonstrated to induce tumorigenesis and invasion. 72 The crosstalk between the MAPK pathway and other signals (such as ErbB3, PI3K/AKT/mTOR, JAK/STAT) is thought to drive human oncogenesis and promote tumor progression [73][74][75][76] (Fig. 2).
In HNC, MAPK pathway mutations occur in~18% of patient tumors. 73 These mutations predominantly occur in BRAF, HRAS, KRAS, and ERK. 77 The activators and regulators of the MAPK pathway (NF1, fibroblast growth factor receptor 2 (FGFR2), FGFR3, and ErbB3) have also been found. 78 Moreover, almost half of the MAPK pathway mutations in head and neck cancer are activating or drivers of tumorigenesis. 70 When growth factors bind to RAS, a signal cascade is activated. ERK1/2 separates from the RAS/RAF/ MEK/ERK1/2 complex and phosphorylates multiple kinases and transcription factors, such as transformation specific-1 (ETS-1), activator protein 1 (AP-1), nuclear factor kappa-B (NF-κB), and c-Myc. 79,80 In the p38/MAPK pathway, mitogen-activated protein kinase-kinase 6 (MAP2K6) overexpression is associated with radiotherapy resistance and unfavorable prognosis in NPC patients. 81 The protein kinase (PAK1) is supposed to phosphorylate RAF1 at serine 338 and MEK1 at serine 298, leading to MAPK activation. 82 JAK/STAT pathway Upregulation of the Jenus-activated kinase (JAK)/STAT pathway, especially STAT3 and STAT5, is associated with cell proliferation, angiogenesis, tumor immune evasion, therapy resistance and poor prognosis in HNC. [83][84][85][86] STAT3 signaling is an immunosuppressive molecule that assists tumor cell immune escape by increasing the production of cytokines, such as TGF-β1, IL-6, IL-10, and VEGF. 87 Mutations in the JAK/STAT pathway are rare in head and neck cancer, as reported previously 88 (Fig. 2).
Several mechanisms are supposed to activate SATA3 signaling, including RTK (EFGR, VEGFR, Src family kinases (SFK), and JAK), TGF-alpha, alpha7 nicotinic receptor, erythropoietin receptor, Gprotein-coupled receptors (GPCRs), Toll-like receptors (TLRs), and the IL-6 cytokine receptor family. [89][90][91][92] Thereafter, phospho-STAT3 in the cell nucleus promotes the expression of downstream target genes, including cyclin D1, survivin and Bcl-xL, which are involved in tumor cell proliferation, angiogenesis and immune evasion. 85,93,94 Moreover, STAT3 is related to increased expression of immune checkpoints, including PD-L1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Combined therapy with checkpoint inhibitors is supposed to decrease the resistance against them. 95 HGF/MET pathway The HGF receptor, the sole ligand of c-MET, is overexpressed in the tumor microenvironment and plays an essential role in tumorigenesis and EGFR inhibitor therapy resistance in HNC. 96 Mutations in the c-MET gene are rare, while an increase in the MET gene copy number and overexpression of HGF are common in HNC. 97 The HGF/MET pathway is crucial for tumor cell proliferation, angiogenesis, invasion and metastasis in HNC. 98 HGF activates c-MET, which promotes cellular morphogenesis, epithelialmesenchymal transition, and tumor metastasis. 99 Major downstream adapter proteins and kinases in the HGF/MET pathway consist of growth factor receptor-bound protein 2 (GRB2), GRB2associated adaptor protein 1 (GAB1), RAS, RAS-related C3 botulinum toxin substrate 1 (RAC1), PI3K, STAT, son of sevenless (SOS), cellular Src kinase, Src homology domain c-terminal adaptor homolog (SHC), SRC homology protein tyrosine phosphatase 2 (SHP2), p21-activated kinase (PAK), and phospholipase c-γ (PLC). [100][101][102] Moreover, the activation of HGF/MET signaling has been demonstrated to influence the cancer stem cell traits of HNC 103 (Fig. 2).
The HGF/MET pathway is supposed to crosstalk with other signaling pathways, including the PI3K/AKT pathway, MEK/ERK pathway, STAT pathway and Wnt pathway, to promote tumor progression. 104,105 The crosstalk between the HGF/MET pathway and the EGFR pathway and VEGFR pathway contributes to therapeutic resistance. 104 p53/retinoblastoma (RB) pathway TP53, a tumor suppressor gene, is one of the most prominently mutated genes in HNC and is associated with tumor progression, recurrence, and therapeutic resistance. [106][107][108] HPV-positive tumors are associated with p53 degradation, retinoblastoma inactivation, and p16 upregulation, while tobacco-related tumors are more associated with TP53 mutation and downregulation of the p16encoded gene. 109 TP53 mutations mostly occur in early tumorigenesis and are related to HPV-negative HNCs because the p53 protein is degraded by the HPV E6 oncoprotein. 110 TP53 encodes a transcription factor that maintains genomic stability, DNA repair, cell cycle, senescence and apoptosis. [111][112][113] P53 is essential for oncogene activation and DNA damage and repair. 114,115 Murine double minute 2 (MDM2), an E3 ubiquitin-protein ligase, negatively regulates the level of p53. 116 P53 can be activated by cell cycle checkpoint kinases (such as CHK1 and CHK2), which induce cell apoptosis and cell cycle arrest. 113,117 The p53 signaling pathway is shown in Fig. 3.
TP63, a p53 family member, is overexpressed in approximately 80% of HNSCC cases. 118,119 TP63 has two isoforms, TAp63 and ΔNp63. ΔNp63 is the predominant p63 isoform, which is vital for tumorigenesis and progression and senescence suppression and is associated with poor OS in HNC. [120][121][122] P63 is considered a stem cell marker. 123 Moreover, ΔNp63 affects the growth factor signaling pathway and tumor metabolic microenvironment through hyaluronic acid signaling and a transcriptional program. 124 TP73, another p53 family member, also consists of TAp73 and ΔNp73 isoforms. P73 is related to the tumorigenesis, angiogenesis, and progression of multiple cancers, including HNC. [125][126][127] Unlike TP53, the TP73 gene is rarely mutated. 128,129 The TAp73 isoform affects tumor cell apoptosis and growth arrest instead of the ΔNp73 isoform. 130,131 The tumor suppressor activity of TAp73 is restrained. The overexpression of TAp73 suppresses the EGFR promoter, downregulates EGFR protein, and induces cell death in HNC cell lines. 130 More research is needed to illuminate the specific contributions of p73 in cancers. 132 The RB gene is one of the most important tumor suppressor genes (TP53, RB, PTEN), which regulates the cell division cycle. 133,134 RB protein binds to EBV protein E7, which leads to protein degradation with E2F release and infinite cell proliferation. 135 When p53 protein is activated by virus infection or DNA damage, the downstream signal p21 is upregulated, which results in RB-E2F complex (RB, E2F, and dimerization partners) formation and cell cycle gene downregulation. Hence, the p53-p21-RB pathway plays an important role in cell cycle arrest. 133 NF-κB pathway The NF-κB family is a cluster of multipotent dimer transcription factors that are closely related to innate and adaptive immune responses, tumorigenesis, and development. [136][137][138] The NF-kB family consists of up to 15 hetero and homodimeric protein complexes drawn from a pool of five monomers. 139,140 In mammals, the NF-κB family consists of RelA (p65), RelB, RelC, NF-κB1 (p105), NF-κB2 (p100), p50, and p52. Major carcinogens, such as tobacco, alcohol, unhealthy diet, irradiation, and oncogenic viruses, are supposed to activate NF-κB. 141,142 The activation of NF-κB is associated with EBV infection, the immunosuppressive tumor microenvironment, the maintenance of cancer stem cell characteristics and metabolic reprogramming in nasopharyngeal cancer. 143,144 The canonical NF-κB pathway depends on cytokine receptors, including interleukin 1 receptor (IL-1R), tumor necrosis factor receptor (TNFR), and Toll-like receptors (TLR4). 145 The upstream signals of the NF-κB pathway are the receptor-interacting proteins (RIP), TNFR-associated factors (TRAF), the kinase TAK1, the adaptor TRADD and FADD. The NF-κB dimers are combined with inhibitory IκB proteins in the resting state but are activated by IκB kinase (IKK) complex phosphorylation when the stimulus appears. The IKK complex is composed of the active kinases IKKα and IKKβ and the regulatory subunit IKKγ (NEMO). Phosphorylated IκB proteins bind to NF-κB dimers and translocate to the cell nucleus. 146,147 The noncanonical NF-κB pathway is activated by CD40 ligand and lymphotoxin-β (LT-β) and defined as IKKα-mediated p100 phosphorylation with RelB, resulting in p100 processing and p52-RelB complex generation. The NF-κB downstream target genes can promote cell proliferation, survival, apoptosis, migration, cell cycle control, and angiogenesis 148,149 (Fig. 3).
Notch1 plays a vital role in maintaining cancer stem cell characteristics and increasing tumor recurrence and metastasis. 178,179 Loss of Notch can promote tumorigenesis by upregulating delta Np63 in HNC. However, delta Np63 expressed in keratinocytes can impair Notch signaling. 180,181 Notch receptors are overexpressed in HNC samples and activate downstream Fig. 3 The p53 signaling pathway and NF-κB pathway in head and neck cancer. CHK1/2 cell cycle checkpoint kinase 1/2, MDM2 murine double minute 2, CDK cyclin-dependent kinase, RB retinoblastoma, TNF tumor necrosis factor, TNFR tumor necrosis factor receptor, TLR4 Tolllike receptor 4, TRAF TNFR-associated factor, RIP receptor-interacting protein, IKK IκB kinase signal transduction via hairy/enhancer-of-split related with YRPW motif 1 (Hey1). 108,182 Inactivating mutations in the Notch gene are observed in 17-26% of HNC cases. 108 Notch1 mutations are predominant, including missense mutations of functional regions, nonsense mutations of truncated proteins, deletions and frameshift insertions. 183 Other signaling pathways in HNC Vascular endothelial growth factor (VEGF), an angiogenesis factor, is highly expressed in HNC and is vital for neovascularization and related to poor prognosis. [184][185][186] VEGF (VEGF-A) is a member of the platelet-derived growth factor (PDGF) superfamily. The ligands of VEGF signals include VEGFR1, VEGFR-2, VEGFR-3, and the coreceptor neuropilins (NRP-1, NRP-2). 187,188 Activated angiogenesis is related to tumor cell proliferation, migration, metastases, and increased sensitivity to radiotherapy and chemotherapy. 189,190 The gene rearranged during transfection (RET) encodes RET kinase, which is one of the receptor tyrosine protein kinases and is related to the tumorigenesis and progression of thyroid cancer. 191 RET has three isoforms, including RET9, RET43, and RET51. The ligands of the RET receptor belong to the glial cell line-derived neurotrophic factor (GDNF) family, which includes GDNF, neurturin, artemin, and persephin. 192 RET receptor binding to its ligands is dependent on a cofactor, which belongs to the growth factor receptor-alpha (GFRα) family, including GFRα1, GFRα2, GFRα3, and GFRα4. GFRα1-4 bind to the RET ligands GDNF, neurturin, artemin, and persephin, respectively, with high affinity and specificity, thereby forming a binary complex and stimulating RET kinase. 193 The phosphorylation of RET kinase activates multiple downstream pathways, including the MAPK pathway, JAK/STAT pathway, PI3K/AKT/mTOR pathway, and PKC, which are associated with tumor cell proliferation, invasion, migration and survival. 194,195 Mutations and rearrangements of the RET gene are commonly discovered in thyroid cancer. Mutations of the RET gene are usually observed in papillary and medullary carcinomas, which are key factors in tumorigenesis and progression. 196,197 Somatic gene rearrangements and fusions of RET occur iñ 2.5-73% of papillary thyroid carcinomas, which are the most common subtype of differentiated thyroid cancer. The most common fusions of the RET gene are NCOA4-RET and CDCC6-RET in papillary thyroid carcinoma. 198,199 Nuclear factor erythroid 2-related factor 2 (NRF2) pathway activation is related to promoting cellular resistance to oxidative stress, proliferation, xenobiotic efflux, metabolic reprogramming, resistance to chemotherapy and radiotherapy. 200,201 Carcinogens trigger c-MYC-mediated NRF2 activation. Under conditions such as oxidative stress, NRF2 eliminates the Kelch-like ECH-associated protein 1/cullin-3 (KEAP1/CUL3) complex and is translocated into the cell nucleus. In the cell nucleus, NRF2 binds to MAF proteins and antioxidant-responsive elements (AREs) and transactivates downstream targeted genes to promote tumor progression in HNC. [202][203][204] Hence, NRF2 upregulation is positively related to the malignant characteristics of HNC. 205 Moreover, NRF2 has been demonstrated to activate the p53/p21 signaling pathway and influence the cell cycle, whereas p62 can inhibit the NRF2/KEAP1/ CUL3 complex. 206,207 Mutations in the NRF2 pathway are commonly observed in HPV-negative HNC patients, while they are rare in patients with HPV infection 208 (Fig. 4).
The overexpression of Sonic Hedgehog pathway proteins, SMO and GLI, has been demonstrated to be a prognostic marker and is related to chemotherapeutic resistance and anti-EGFR therapeutic resistance in HNC. [214][215][216][217] Moreover, radiotherapy is supposed to induce GLI1 expression mediated by the mTOR/S6K1 pathway, which in turn causes radioresistance 218 (Fig. 4).
In addition, Toll-like receptors (TLRs), especially TLR2/3/5, are highly expressed in HNC and are closely related to poor prognosis. [219][220][221] Engagement of TLR3 is supposed to activate TRIF and trigger downstream signaling to activate the NF-κB pathway. 222 TLR2/5 activation is mediated by the c-jun N-terminal kinase-related pathway, PI3K/AKT pathway, and NF-κB pathway. 223 Overall, multiple signaling pathways are involved in regulating the tumorigenesis and progression of HNC. Among them, the EGFR pathway plays a critical role in HNC and can be activated by EGF, and downstream pathways include the PI3K/AKT, MAPK, JAK/ STAT, and other signaling pathways. Moreover, the EGFR pathway can interact with other RTKs, including HER, MET, IGF-1R, VEGFR, and RET, to synergistically regulate the occurrence and development of HNC. TP53 is an important tumor suppressor gene. The p53 family is involved in HNC tumorigenesis and development, regulating the cell cycle, genomic stability, DNA repair, and apoptosis. NRF2 can activate the p53 pathway. In addition, the NF-κB pathway is also important in HNC and is supposed to crosstalk with the STAT, PI3K, and p53 pathways to promote tumor prognosis in HNC. The Notch signaling pathway is involved in HNC radiation resistance and can be activated by PI3K signaling, which can be suppressed by PTEN. Hence, the crosstalk among these signaling pathways is complex in tumorigenesis, progression, and therapy resistance in HNC.

TARGETED THERAPY IN A PRECLINICAL MODEL IN VIVO
Vivo models can be separated into spontaneous, induced, transplantation, and transgenic models. Transplantation models include subcutaneous tumor models, orthotopic tumor models, and patient-derived tumor xenografts (PDXs). In addition to the traditional animal model, novel animal models have been used in developing antitumor drugs in HNC. 224,225 PDXs are established by patient-derived tumor tissue directly implanted in immunodeficient mice without prior management or planting in wells, which is a new method for evaluating novel therapies. 226 The construction of a PDX model also provided a novel approach for identifying suitable alterations in tumor tissue to find promising treatment methods.
HPV is the most important oncogenic factor in HNSCC. According to the common evidence of the subtypes of HNSCC, one is HPV-related, and the other is alcohol, tobacco or oral trauma-related: HPV-positive and HPV-negative. 227 The clinical and biological characteristics are opposite. 228 As mentioned previously, the PDX model provides a novel approach for identifying suitable alterations in tumor tissue to find promising treatment methods. [229][230][231][232] Recently, this model has been considered an ideal preclinical model for investigating targeted drugs. These PDX models maintained the genetic background in immunodeficient mice. According to the pathology, genes, and cells of tumors, the response in mice can simulate that in humans. [233][234][235] The response of tumors with HPV or not brother the response of treatment regimens. [236][237][238][239] The PDX model can reflect the status of HPV. HPV-negative tumors have been shown to be associated with favorable survival. [240][241][242][243] Previous results in PDX models were identified in clinical trials, which showed that they have similar antitumor properties. PDX models can maintain the structure of tumors and histological characteristics, although it is still debatable how long the microvessel structure from human tissue is maintained. 244,245 Some studies claimed that prostate tumors derived from humans maintained their microvasculature after transplantation into mice and that the vessel density of the tumor was enhanced after weeks of transplantation. 246,247 In contrast, a study reported that the vessel density of renal cell carcinoma decreased after transplantation. 246,247 The difference after tumor implantation in nude mice may explain why some VEGFR inhibitors obtained resistance in the mouse model. Microvasculature variation may also serve as an explanation for the resistance to antiangiogenic drugs. In addition, precise research including one hundred fifty PDX mouse models assessed angiogenic phenotypes to develop a system to select suitable patients to receive targeted therapy, which may help overcome tumor heterogeneity and predict prognosis. 248 A comprehensive understanding and inhibitor direction for targeting signaling pathways in preclinical HNC treatment are shown in Fig. 5.

EGFR inhibitors
Cetuximab is the only FDA-approved EGFR-targeted therapy in HNSCC. [249][250][251] Many compounds targeting EGFR have been developed in preclinical models, with promising response and tolerability profiles. Referencing the current knowledge that multimodality treatment provides new hope for aggressive HNSCC, many studies have investigated the response of combination treatment regimens, either with conventional surgery, radiotherapy and cytotoxic chemotherapy, or in novel precision medicine targeted or immunotherapy combinatorial regimens. In xenograft tumor tissues, EGFR overexpression promoted the radiation therapy response in HPV-positive HNSCC by attenuating DNA damage repair and HPV E6 decrease. 252 Early in 2001, the effect of cetuximab was investigated in squamous cell carcinoma in vivo, which also enhanced the efficacy of radiotherapy. 253,254 Xenograft models revealed that EGFR inhibitor monotherapy led to partial and transient tumor regression. The levels of caveolin-1 and Sox-2 in PDX tumors acted as predictive biomarkers of the cetuximab response in HSNCC cells. The accuracy reached 88% according to the cetuximab response. 255 The combination of cetuximab with either gefitinib (Iressa, and ZD1839, EGFR inhibitor) or erlotinib (Tarceva, and OSI-774, EGFR inhibitor) was investigated in HNSCC. More profound tumor regression and regrowth delay were observed in an in vivo model administered the combination of cetuximab and gefitinib or erlotinib. 256 The combination of cetuximab with other treatment regimens was further investigated in HNSCC models.
CP-358,774 is a novel potential and selective inhibitor of EGFR and inhibits EGF-mediated mitogenesis in cancer cells. CP-358,774 in combination with cisplatin demonstrated a better response than cisplatin monotherapy with no detectable decrease in body weight or adverse effects. 257 GW2016 is an effective inhibitor of the ErbB-2 and EGFR tyrosine kinase domains that is undergoing development. ZD6474 is a dual inhibitor of VEGFR-2 and EGFR tyrosine kinase. The efficacy of ZD6474 in HNSCC xenografts was detected. In vivo, ZD6474 inhibited tumor growth via apoptosis and antiangiogenic activity. 258 Vandetanib (Zactima) is another kind of inhibitor of VEGFR-2, RET, and EGFR. Adenoid cystic carcinoma accounts for 1% of all HNC cases and can be controlled by surgery combined with radiotherapy. No efficient therapeutic compounds have been developed. In the parotid glands of a mouse model, vandetanib was well tolerated and inhibited the mean tumor volumes. 259 Gefitinib is a selected EGFR inhibitor that inhibits signal transduction to attenuate malignant neoplasm cell growth and proliferation. Gefitinib has demonstrated potential therapeutic activity in HNSCC in vivo by blocking basal EGFR-mediated mitogenic signaling. 260 Combination therapy with paclitaxel and EGFR inhibitors improved prognosis in a mouse model of oral cancer by promoting cell apoptosis. VEGF inhibitors combined with EGFR inhibitors and radiotherapy resulted in a significant response in the HNC orthotopic model. 261 The response to combination therapy regimens with EGFR inhibitors in HNSCC was remarkable, while the clinical efficacy of treatment with EGFR inhibitors remained insufficient. In addition to an insufficient response rate, nearly all patients with clinical response finally developed resistance status after approximately ten months of EGFR inhibitor treatment, which indicated that during EGFR inhibitor treatment, some alterations led to the occurrence of resistance. In HN4 and HN6 cell xenograft models, the nerve growth factor (NGF)-TrkA axis induced epithelialmesenchymal transition (EMT) and mediated resistance to EGFR inhibitors. 262,263 Dacomitinib (EGFR, ErbB-2, and ErbB4, irreversible inhibitor) was efficient in inhibiting the tumor volume of HNSCC and acted as a radiosensitizing agent in HNSCC. Dacomitinib treatment enhanced the effect of radiotherapy. 264 AC480 is a novel pan-HER (EGFR and HER2) inhibitor that alone cannot promote tumor cell apoptosis. However, combination with radiotherapy could enhance its effect. The tumor size of HNSCC xenografts in vivo was significantly reduced after AC480 plus radiation. 265 VEGF inhibitors Bevacizumab, sorafenib, and sunitinib are the most common VEGF inhibitors used in targeted therapy. 266 Bevacizumab is a monoclonal antibody that inhibits angiogenesis in tumors. 267 The antitumor effect of bevacizumab has been investigated in preclinical tumor models. Bevacizumab successfully inhibited HNSCC growth and had a promising effect on inhibiting HNSCC. Extracellular matrix metalloprotease inducer (EMMPRIN and CD147) is a membrane-bound glycoprotein observed on the membrane of cancer cells. 268 CD147 overexpression was shown in distinct types of tumors, including HNSCC, hepatocellular carcinoma, gastric cancer, thyroid carcinoma, cervical adenocarcinomas, bladder cancer, and ovarian cancer. 269,270 The overexpression of CD147 in HNSCC xenografts promoted tumor growth and facilitated the production of VEGF. 271 CD147 also acted as a predictive effector in predicting the response to bevacizumab in an HNSCC xenograft model. 272 Although bevacizumab could not inhibit the proliferation of HNSCC cell lines, bevacizumab demonstrated dramatic anticancer efficiency in an HNSCC xenograft model. Bevacizumab plus paclitaxel led to a remarkable effect in HNSCC tumors compared with the single compound effect. 273 The HNSCC xenograft model benefited from bevacizumab in combination with cisplatin. However, cetuximab treatment alleviated the synergistic effect of bevacizumab and cisplatin. 274 Cetuximab treatment in combination with other regimens needs further exploration. In addition to HNSCC, bevacizumab had a potential effect in a mouse model of medullary thyroid carcinoma (MTC) and anaplastic thyroid carcinoma. Bevacizumab and EGFR inhibitor monotherapy or in combination blocked angiogenesis in tumors and inhibited tumor growth. Both regimens were superior to doxorubicin treatment. 275 Pretreatment with bevacizumab promoted radiotherapy in MTC with moderate adverse effects. 276 Lenvatinib is a novel dual inhibitor targeting VEGFR-2 and FGFR1 that demonstrates robust anticancer efficiency in PDXs and humanized mice, which is equivalent to bevacizumab. 277,278 AEE788 is a specific kinase inhibitor targeting VEGFR and has a potential effect on EGFR. AEE788 alone and in combination with paclitaxel significantly inhibited the proliferation of tumors in the tongues of athymic nude mice. 279,280 Apatinib is a novel selective VEGFR-2 inhibitor that inhibits the proliferative ability of thyroid carcinoma and squamous cell Comprehensive understanding and inhibitor direction for targeting signaling pathways in preclinical HNC treatment. EGFR epidermal growth factor receptor, EGF epidermal augmentum factor, MET mesenchymal-epithelial transition factor, JAK Janus-activated kinase, STAT signal transducer and activator of transcription, AKT serine/threonine-specific protein kinase, mTOR mammalian target of rapamycin, CDK cyclin-dependent kinase, VEGF vascular endothelial growth factor, mAb monoclonal antibody, RET rearranged during transfection, p phosphorylation carcinoma in vivo and vitro and has been identified in multiple studies. 281,282 In a tumor xenograft model, mice benefited from either apatinib alone or apatinib in combination with cytotoxic drugs. 283 Tumor angiogenesis was also suppressed after apatinib treatment. The outcomes demonstrated that apatinib inhibited cancer growth compared with the untreated model group.
Apatinib combined with radiation therapy demonstrated a stronger effect in inhibiting tumors than either apatinib treatment alone or radiation therapy alone. The tumor partial oxygen pressure, VEGFR-2-positive cells, and CD31-positive cells in the combination group were lower than those in the single regimentreated group, which demonstrated that angiogenesis in tumors was blocked. 283 SCH772984 is an ERK inhibitor and has no effect on tumor growth. Apatinib combined with SCH772984 exhibited a greater inhibitory effect on tumor growth of oral squamous cell carcinoma in vivo, which revealed that inhibition of ERK promoted the anticancer efficiency of apatinib in vivo. 284 VEGF inhibitors, including ONC201, cabozantinib (XL-184), linifanib (ABT-869), sunitinib, motesanib, pazopanib, axitinib (AG-013736), and PTK787/ZK 222584, all had promising effects in the HNC model. [285][286][287][288][289] FGFR inhibitors BGJ398 is a pan-FGFR inhibitor administered orally that mainly targets FGFR1-3. The effect of BGJ398 in HNSCC was closely related to the expression of FGFR in vivo. 290 Furthermore, the selective FGFR inhibitor PD173074 alone also resulted in a remarkable response in an HNSCC xenograft model. 291 AZD4547 is another novel inhibitor with a potent ability to inhibit FGFR1, FGFR2 and FGFR3, which could promote the effect of radiotherapy in an HNSCC patient-derived xenograft model. 292 PI3K/Akt/mTOR pathway inhibition PI3K inhibitors. PI3K inhibitors alone do not exhibit a remarkable efficacy like an EGFR tyrosine kinase inhibitor and only selected tumor benefits from them. Human HNSCC xenografts with PIK3CA mutations exhibited susceptibility to therapy with PI3K inhibitors.
BYL719 is a kind of PI3K inhibitor that demonstrated a significant antitumor effect on HNSCC in xenografts. HNSCC patients could benefit from BYL719 inhibitors, and depletion of MYC, p53 mutation, or YAP especially potentiates patients. 293 Combining BYL719 with KTN3379, a monoclonal antibody targeting HER3, enhanced the suppression of HNSCC in vivo. 294 However, some HNSCC cells with PI3K-independent activation were resistant to PI3K-independent activation. The AXL-EGFR interaction mediated the process and promoted the antitumor effect of the PI3Kα inhibitor BYL719. 295,296 Ribociclib is a specific CDK4/6 inhibitor that showed a synergistic effect in combination with BYL719 in nonkeratinizing NPC. 297 IGF2 inhibitors enhanced the efficacy of BYL719 and taselisib (GDC0032) for the treatment of HPV-positive HNSCC. A PI3K inhibitor also showed promising anticancer effects in cetuximab-resistant oral squamous cell carcinoma. 298 BKM120, a PI3K inhibitor, inhibited HNSCC cell proliferation in vivo. 299,300 Among the 353 tested cell lines in mouse xenografts, BKM120 particularly inhibited cancer cells with somatic PI3Kα alternations. BKM120 combined with cetuximab and irradiation significantly inhibited orthotopic xenograft tumors of HNSCC, which provided a rationale for clinical treatment. 301 Both BKM120 and BYL719 promoted radiosensitive effects in an HNSCC xenograft model. PX-866 also had anticancer efficacy with PIK3CA alterations. 302,303 Compared with radiation monotherapy, taselisib, a PI3K inhibitor, monotherapy improved the inhibition of cancer cell proliferation. Taselisib plus radiation therapy completely inhibited cancer cell growth, while no significant difference between gross tumor volume was observed at the beginning and end of therapy after three months. 58,304 LY294002 and copanlisib (BAY 80-6946) were also developed in cancer cell xenograft models. [305][306][307] Moreover, patients who responded to PI3K inhibitors acquired drug resistance over time. Few studies have observed acquired resistance to PI3K inhibitors in HNSCC. In HNSCC PDXs that develop resistance to PI3K inhibitors, MAPK activation was detected in the tissue. Inhibiting MAPK activity could resensitize drug-resistant cells to PI3K inhibitors. 308 AKT inhibitors. AKT overexpression in malignant tumor cells demonstrated a stronger response to perifosine than AKTdownregulated cells. 309,310 The response in tumor cells is related to the phosphorylation of AKT. Perifosine, an AKT inhibitor, promoted cell growth, and apoptosis and blocked the cell cycle, which provided considerable insight into tumor treatment. 311,312 In HNSCC xenografts, perifosine combined with radiotherapy completely inhibited tumor proliferation and prolonged tumor survival and regression via apoptosis. MK-2206, a novel AKT inhibitor, plus cisplatin also showed a synergistic effect in HSNCC cells. 313 The ATP-competitive inhibitor ipatasertib (GDC0068) demonstrated significant anti-proliferative effects in mouse xenografts, and the effect in PTEN mutation tumors or with PI3K alterations was enhanced. The AKT inhibitor capivasertib (AZD5363) inhibited AKT activation and resensitized saracatinibresistant HNSCC cells to saracatinib. 314 Capivasertib in combination with saracatinib inhibited tumor proliferation more efficiently than either agent in xenografts. mTOR inhibitors. mTOR inhibitors have been proven to improve the efficacy of chemotherapy and radiotherapy without increasing adverse effects by preventing lactate production and inhibiting HNSCC cell proliferation. 315 In an orthotopic xenograft model of HNSCC, temsirolimus, a potent mTOR inhibitor, exhibited effects on tumor growth. Temsirolimus plus an anti-EGFR inhibitor had a synergistic anticancer effect. 316 Both temsirolimus and RAD001 treatment showed significant tumor shrinkage, and mTOR activation was inhibited in HPV-positive oral and cervical squamous cell carcinoma (SCC) xenografts. Phosphorylation of its downstream effectors pS6 and pAKT (S473) was observed to be inhibited. 37 RAD001 also prevented tumor growth of cells with HPV-negative TP53 mutation in vivo through autophagy activity. 317 The novel mTOR inhibitors CZ415, AZD8055, OSI-027 (ASP4876), and CC-223 monotherapy inhibited HNSCC cell growth in vivo. 318-320 mTOR monotherapy and combination with other treatment regimens all demonstrated promising effects on the HNSCC xenograft model. PF-04691502 is an ATP-competitive, dual inhibitor of PI3K and mTOR administered orally. PF-04691502 has produced significant radiosensitization in nonmetastatic HNSCC xenografts. 321 The study demonstrated that radiosensitization in all HNSCC cell lines was identified regardless of p53, and treatment with PF-04691502 downregulated radiosensitization in normal fibroblasts compared with tumor cells. When the PI3K/AKT/mTOR survival pathway was activated by radiotherapy, PF-04691502 facilitated the efficacy of radiotherapy by inhibiting phosphorylation of effectors in the pathway in HNSCC xenografts, similar to other inhibitors in the PI3K/AKT/mTOR pathway. 322 Antitumor activity was also observed in HNSCC xenografts with alterations in PIK3CA after treatment with the dual PI3K-mTOR inhibitor PF-5212384, and the activity was promoted by the MEK inhibitor PD-0325901. 323 BGT226 significantly inhibited tumor growth in HNSCC xenografts. 324,325 The PI3K/Akt/mTOR pathway has been widely developed in HNSCC. Many inhibitors have been investigated in preclinical studies.
c-MET inhibitors BPI-9016 M is a c-MET inhibitor that inhibits tumor growth by promoting tumor cell apoptosis and facilitating DNA damage. Treatment with BPI-9016 M promoted the radiosensitization of tumors in vivo. The volume of esophageal squamous cell carcinoma tumor xenografts was significantly reduced in the combination group compared with each regimen alone. 326 In a xenograft model, the c-MET inhibitor tepotinib also enhanced the effect of radiotherapy. In addition to radiotherapy, the c-MET tyrosine kinase inhibitor PF-2341066 enhanced the effect of cisplatin in HNSCC. 327 Not all c-MET inhibitors in combination with other regimens had a synergistic effect. Capmatinib (c-METspecific inhibitor) plus pitavastatin (HMGCR inhibitor) synergistically inhibited tumor growth and served as a novel treatment regimen in oral and esophageal cancer. However, pitavastatin combined with other c-MET inhibitors (crizotinib or MGCD-265) had no synergistic action on tumors in vivo compared with capmatinib plus pitavastatin. 328 The results showed that pitavastatin and capmatinib were some of the best combination approaches for inhibiting tumors in vivo, which is worth exploring in clinical trials. The multiple AXL/MET/VEGFR inhibitor glesatinib combined with the MEK inhibitor trametinib also exhibited a remarkable effect in inhibiting tumors. Trametinib as a single compound decreased the tumor volume up to 72% compared with 91% in the combination group. The outcomes demonstrated that mice treated with glesatinib in combination with trametinib showed a promising increased magnitude and durability of response. 329 The novel c-MET inhibitor TR1801-ADC (cMettargeted "third-generation" ADC), PF-2341066 (selective c-MET inhibitor), cabozantinib (XL-184), and PHA665752 showed promising effects in HNC in vivo. [330][331][332] RAF inhibitors RAF inhibitors have a vital role in treating thyroid cancer, and the efficiency of RAF inhibitors is associated with BRAF status. Vemurafenib, a BRAF kinase inhibitor, has remarkable effects in thyroid cancer, but drug resistance results in vemurafenib treatment failure. LY3009120 (pan-RAF inhibitor) helped overcome vemurafenib resistance. 333,334 PLX4720, a highly selective B-Raf (V600E) inhibitor, significantly attenuated tumor aggression in vivo in thyroid cancer with the B-Raf (V600E) mutation, which also contributed to drug resistance. 335,336 A tyrosine kinase inhibitor (ponatinib) combined with PLX4720 showed amazing synergistic action in xenograft models of B-Raf (V600E) thyroid cancer, which also contributed to overcome PLX4720 resistance. 337 The c-MET kinase inhibitor PF-04217903 could enhance the effect of the MEK/RAF inhibitor CH5126766 in murine anaplastic thyroid cancers, which provides new help to patients. 338 B-Raf (V600E) inhibitor also acted as a radiosensitizer in B-Raf (V600E)-mutant thyroid cancer cells. 339 RAF inhibitors combined with PI3K inhibitors or MEK1/2 inhibitors all exhibited promising effects in thyroid cancer in a mouse model. 340,341 MEK and ERK inhibitors MEK inhibitors, such as U0126, trametinib, selumetinib, AZD6244, and PD-0325901, demonstrated promising effects in thyroid cancer and squamous carcinoma in HNC in a mouse model. The MEK inhibitor trametinib also resensitized saracatinib-cisplatin HNSCC cells to cisplatin in an orthotopic xenograft model. PD-0325901 enhanced the radiosensitization of HNSCC in vivo. 342,343 The results offer new insight into overcoming chemoresistance in preclinical HNSCC models and contribute to the further use of MEK inhibitors in clinical research. 344 The MEK inhibitor selumetinib has been approved in advanced differentiated thyroid carcinoma. 345 However, some tumors develop MEKi resistance, but the mechanism remains unknown. After MEK inhibitor resistance animal models were established successfully, RTK and SHP2 were observed to be active. SHP2 inhibitor combined with AZD6244 significantly inhibited the tumor volumes and weight compared with the control group, which was superior to singleagent treatment. AZD6244 could also enhance the efficiency of the selective RTK inhibitor tipifarnib in HRAS-driven dedifferentiated thyroid cancers in vivo. 346 In PDX models of thyroid cancer with different KRAS, BRAF, and NRAS mutations, the MEK inhibitor selumetinib combined with the MDM2 inhibitor KRT-232 showed a remarkable effect. Combination treatment prolonged the survival of mice via MAPK signaling pathway blockade. 347 Although patients could significantly benefit from lenvatinib (E7080, multitarget inhibitor, mostly for VEGFR-2 (KDR)/VEGFR3 (Flt-4)), the adverse effect of lenvatinib was not tolerated in patients with radioiodine-refractory thyroid cancer. 348 Lenvatinib plus the MEK inhibitor selumetinib (AZD6244) promoted the anticancer effect in mice with anaplastic thyroid cancer. Combining selumetinib with an FGFR3 inhibitor (PD173074) significantly reduced the tumor volumes and weight in HNSCC xenografts. 349 DEL-22379, a relatively specific ERK inhibitor, showed remarkable anticancer efficiency in BRAF-mutant anaplastic thyroid cancer in vivo and was a candidate target for cancer therapy. 350 Notch inhibitors AL101 (osugacestat) is a potent γ-secretase inhibitor that blocks the activity of all four Notch receptors. In an adenoid cystic carcinoma xenograft model with Notch1 mutations, the tumor volumes and body weight were reduced by AL101, which provided fairly broad therapeutic prospects in adenoid cystic carcinoma. 351 JAK/STAT inhibitors AZD1480 is an inhibitor targeting JAK1/JAK2 that has been investigated in several tumor models. In PDX models from two independent HNSCC patients, treatment with AZD1480 significantly decreased tumor volumes and weight by decreasing pSTAT3 Tyr705 phosphorylation. 86,352 In accordance with the promising effect of AZD1480 in HNSCC, another JAK2/STAT3 inhibitor, AG490, was tested in a HNSCC transgenic mouse model, which not only inhibited angiogenesis by suppressing the VEGF receptor but also decreased myeloid-derived suppressor cells. 353 AG490 further suppressed metastasis and tumor cell proliferation in oral squamous cell carcinoma in vivo. 354 The JAK/STAT signaling pathway was active after radiotherapy, which may contribute to radiotherapy resistance to reduce the antitumor effect. NVP-BSK805, an inhibitor of JAK2 kinase, promoted DNA doublestrand breaks to block the cell cycle and suppress DNA repair to enhance the radiosensitizing effect in esophageal squamous cell carcinoma in vivo. The results demonstrated an attractive prospect of NVP-BSK805 in HNSCC. 355 CDK4/6 inhibitors The effect of CDK4/6 inhibitors is a hotpot in HNSCC. [356][357][358][359][360] Abemaciclib is the first CDK4/6 inhibitor that has been widely investigated. In vivo, abemaciclib inhibited cancer cell growth compared with the model group without inducing tumor recurrence. The combination of mTOR inhibitors with abemaciclib enhances its anticancer effect. 361 PD-0332991 is a specific inhibitor of CDK4/6 that has demonstrated potent anticancer effects in HNSCC. The metastasis of esophageal squamous carcinoma could also be suppressed by PD-0332991. The mentioned results provide promising insight into the use of PD-0332991 in clinical trials. 362 SHR6390 is another new inhibitor specifically inhibiting CDK4/6. SHR6390 suppressed tumor cell growth in vitro and suppressed tumor growth in PDX models. [363][364][365] This kind of inhibitor suppressed RB phosphorylation and blocked the cell cycle at G1 in vivo. The combination of SHR6390 with paclitaxel or cisplatin had synergistic action in inhibiting tumor growth in vivo. 365 The expression of cdK4/6 was related to the prognosis of tumors.
Ribociclib (LEE011) is a selective CDK4/6 inhibitor used in aggressive thyroid cancer via oral administration in a PDX tumor model. 366 Ribociclib promoted the sensitivity of radiation-resistant esophageal cancer cells by inhibiting YAP1 expression. The combination of ribociclib with radiation overcame radiotherapy resistance in HNSCC in vivo. 367 Ribociclib has a remarkable influence in HPV-negative HNSCC models. However, ribociclib had no significant efficacy in HPV-positive HNSCC models. In patientderived tumor xenograft models, the response to ribociclib is closely correlated with retinoblastoma protein production. 368 The combination of cetuximab and ribociclib showed no synergistic effect in the HNSCC model, but the administration of ribociclib promoted sensitivity to CDK4/6 inhibitors in cetuximab-resistant HPV-negative PDX models. 359 Palbociclib could block the cell cycle at G1 phase by targeting CDK4/6. In Epstein-Barr virus-positive HNSCC, palbociclib demonstrated promising effects and decreased the Epstein-Barr virus titer. 369 In oral squamous cell carcinoma patient-derived xenografts, palbociclib showed limited efficiency. The administration of combination palbociclib with cetuximab was investigated in preclinical studies, but the effect needs further exploration. 370 CDKN2A/2B mutation in PDX models acted as a predictive biomarker in predicting palbociclib response. PDX of esophageal squamous cell carcinoma with CDKN2A/2B loss was more sensitive to CDK4/6 inhibitors than those with wild-type CDKN2A/2B. 371 Palbociclib has shown a promising effect in HNSCC, and the adverse effect of palbociclib is well tolerated. Some drugs have synergistic effects in treating HNSCC. To further explore the synergistic effect of palbociclib plus other drugs, etuximab (EGFR inhibitor), PF-04691502 (PI3K inhibitor), and carboplatin were evaluated, but the efficiency of those regimens was not satisfactory. Trametinib, a MEK inhibitor, has shown a remarkable influence in suppressing HNSCC in vivo. The combination of palbociclib and trametinib was developed in Detroit 562 cells in immunodeficient mice. The tumor weight and volume were reduced by the combination therapy compared with either monotherapy. No significant profiles were observed after palbociclib and trametinib combination treatment. 372 Palbociclib and SAHA combination therapy also exhibited a synergistic effect in suppressing NPC growth. 373 LY2835219, a selective CDK4/6 inhibitor, inhibited tumor cell proliferation and blocked the cell cycle by suppressing CDK4/6-dependent Ser780 phosphorylation, which showed a potent effect in treating HNSCC. The combination of LY2835219 with a mTOR inhibitor significantly suppressed HNSCC tumor growth in vivo. 361,374 RET inhibitors RPI-1, a novel 2-indolinone RET tyrosine kinase inhibitor, has shown promising results in sporadic papillary thyroid carcinomas with frequent RET alterations in vivo. 375 The therapeutic effect was quite immediate, and tumor proliferation was efficiently controlled, with the tumor weight reduced to 20% of the control group. RPI-1 also has remarkable efficacy in MTC xenografts by facilitating tumor cell apoptosis and inhibiting angiogenesis. 376 The results demonstrated that RET oncogene activity is closely related to the maintenance and survival of MEN2A-type MTC, which contributed to the further administration of RPI-1 in treating thyroid cancer with RET alternation.
NVP-AST487 is another novel RET tyrosine kinase inhibitor that can also inhibit KDR, Flt-4, Flt-3, c-Kit, and c-Abl. 377 The effect of NVP-AST487 was identified in vivo in medullary thyroid cancer with oncogenic RET. The efficiency of NVP-AST487 was observed to be dose-dependent on RET expression. In the mice treated with 50 mg/kg, the tumor volumes, weight and RET production all exhibited a dramatic decrease. Some multiple target inhibitors, vandetanib and XL-184, were reported to target RET in addition to EGF-R2, VEGF-R3, and EGFR. 285,378,379 As discussed above, vandetanib showed remarkable effects in adenoid cystic carcinoma, and XL-184 also presented promising results and a moderate effect in HNC by targeting other receptors. 380 Novel inhibitors JNK inhibitors, such as SP600125 and AS601245, have been evaluated in preclinical studies in vivo. 381 The focal adhesion kinase (FAK) inhibitor defactinib also exhibited an excellent effect in HNSCC. In addition, dual inhibitors have been developed and investigated in xenograft models. Bosutinib (SKI-606) is a secondgeneration tyrosine kinase inhibitor that acts as a dual inhibitor of Src and Abl. Bosutinib demonstrated a remarkable effect in suppressing tumor growth in vivo. 382 Table  1). 25 Except for acneiform rash and infusion reactions, no significant difference was observed in the incidence of other severe adverse effects between groups, including mucositis, nausea, and radiation dermatitis. These promising results allowed the FDA approval of cetuximab in combination with radiotherapy for the treatment of LA-HNSCC in 2006. The 5-year follow-up of this study also supported the superiority of the combination strategy over radiotherapy alone. 385 Moreover, cetuximab-treated patients who experienced serious rash exhibited better survival than those with no or low-grade rash. 385 However, this does not necessarily mean that cetuximab plus radiotherapy can be an effective and safe replacement for the standard chemoradiation composed of cisplatin and radiotherapy. Head-to-head comparison studies indicated that cetuximab + radiotherapy is inferior to the standard chemoradiotherapy (cisplatin + radiotherapy) strategy, with reduced treatment compliance, comparable efficacy outcomes, and increased toxicities. [386][387][388] In HPV-positive patients, the addition of cetuximab to radiotherapy showed lower local disease control and shorter survival outcomes than the addition of cisplatin. 389,390 Cetuximab also brings no additional benefits when combined with cisplatin plus radiotherapy. 391,392 Therefore, cisplatin remained the first-choice radiosensitizer in all eligible patients, especially for those with HPV infection.
Induction chemotherapy (ICT) with taxanes, cisplatin, and 5-FU (TPF) before receiving chemoradiotherapy or radiotherapy resulted in increased tumor responses and reduced failure in local control and distant metastasis in LA-HNSCC. 393,394 Whether the addition of cetuximab to ICT regimens brings more benefits than drawbacks remains controversial. In several phase 2 clinical trials, adding cetuximab to the TPF regimen resulted in tolerable and long-term control of LA-HNSCC, especially in HPV-negative cases. 395,396 However, the head-to-head DeLOS-II trial indicated that the addition of cetuximab to the TPF ICT regimen (TPF-C) showed no superiority in survival outcomes over TPF alone. 397 Nevertheless, cetuximab can be considered an effective and tolerable substitute for 5-FU in this regime, with a comparable overall response rate (ORR) and OS and slightly fewer serious adverse effects. 398 Cetuximab, paclitaxel, and carboplatin (PCC) is an alternative ICT regimen that is safe and could help induce a strong local response and promising survival. 399,400 Other monoclonal antibodies of EGFR are still under intensive investigation, including panitumumab, nimotuzumab, zalutumumab, etc. Similar to cetuximab, panitumumab cannot replace  Targeted therapy for head and neck cancer: signaling pathways and. . . Li et al. cisplatin when combined with radiotherapy for LA-HNSCC according to the results from the CONCERT-2 401 and HN.6 402 trials. Panitumumab plus radiotherapy showed no advantage in improving the local control rate, survival time, and quality of life in treated patients compared with cisplatin plus radiotherapy. Meanwhile, adding panitumumab to the standard chemoradiotherapy strategy failed to provide any benefit, 403 making panitumumab an unsuitable choice for LA-HNSCC patients. The addition of nimotuzumab to radiotherapy with or without cisplatin provided long-term survival benefits for up to five years and improved the complete response rate in LA-HNSCC patients. 404,405 In a phase 3 clinical trial involving 536 LA-HNSCC patients, nimotuzumab plus cisplatin and radiotherapy significantly improved the locoregional control rate and PFS without negatively impacting the quality of life. 406,407 Except for a higher incidence of mucositis, other adverse effects of grade 3 or more were similar with or without nimotuzumab. 406 The promising results strongly supported the addition of nimotuzumab to LA-HNSCC patients who are treated with cisplatin and radiotherapy. Another monoclonal antibody, zalutumumab, extended the survival time from 8.4 to 9.9 weeks in recurrent or metastatic (R/M) HNSCC patients who had failed platinum-based chemotherapy. 408 Meanwhile, moderate-to-severe skin rash during zalutumumab treatment was related to superior OS, independent of HPV infection and p16 status. 409 Some small molecular inhibitors of EGFR are also under investigation for the management of HNSCC, including selective inhibitors (e.g., gefitinib, erlotinib) and dual-target inhibitors (e.g., afatinib, lapatinib, and dacomitinib). Gefitinib is an orally administered selective inhibitor of EGFR. When administered fluorouracil, hydroxyurea, and radiotherapy, gefitinib demonstrated a strong complete response rate and favorable survival outcomes (4-year OS: 74%; PFS: 72%) after a median follow-up of 3.5 years in LA-HNSCC patients. 410 Lapatinib monotherapy also showed evidence of clinical activity with an ORR of 17% in LA-HNSCC patients compared with placebo. 411 Lapatinib plus chemoradiotherapy is safe and induces a high complete response rate and long median PFS in p16-negative LA-HNSCC patients. 412 However, the addition of lapatinib to chemoradiotherapy, followed by lapatinib maintenance brought additional toxicity with limited efficacy in patients with high-risk HNSCC after surgery. 413 Therefore, lapatinib is unsuitable for long-term maintenance treatment.
EGFR inhibition in recurrent or metastatic head and neck cancer (R/ M-HNC). Cetuximab monotherapy was well tolerated and induced a response rate of 13% in R/M-HNSCC patients. 414 Combining cetuximab with cisplatin contributed to a significant improvement in ORR (26% vs. 10%) but failed to prolong survival for R/M-HNSCC patients when compared with cisplatin monotherapy. [415][416][417] In the landmark EXTREME study, cetuximab combined with platinum (cisplatin or carboplatin) and fluorouracil (PCF) resulted in improved median PFS (5.6 months vs. 3.3 months), OS (10.1 months vs. 7.4 months), and response rates (36% vs. 20%) compared with chemotherapy alone. 26 This led to the approval of cetuximab in combination with platinum-based therapy with fluorouracil as the first-line treatment for R/M-HNSCC patients. However, this standard of care regime has some disadvantages in clinical use, including the requirement of hospitalization to ensure proper hydration and continuous infusion of fluorouracil and severe toxicities such as nausea and anorexia. To improve the feasibility and reduce the adverse effects, cetuximab is under investigation for combining different chemotherapeutics in the treatment of R/M-HNSCC. For instance, cetuximab combined with paclitaxel, 418 docetaxel and cisplatin, 419 or paclitaxel and platinum showed promising activity and tolerability as first-line treatment in R/M-HNSCC patients. 420,421 The cetuximab, carboplatin, and paclitaxel (PCC) regimen induced an ORR of 40%, median OS of 14.7 months, and median PFS of 5.2 months, comparable to those reported with PCF treatment. 26,420 Further studies involving more patients are needed to confirm the efficacy of PCC and compare it with PCF. In the GORTEC 2014-01 TPExtreme phase 2 study, although a combination of cetuximab, docetaxel, and cisplatin (TPEx) showed no significant improvement in survival outcomes in R/M-HNSCC patients when compared with the PCF regimen, it showed a significantly better safety profile, with fewer people experiencing grade 3 or worse adverse effects (81% vs. 93%). 422 Therefore, the TPEx regime can be a reliable alternative to the PCF regime in the first-line treatment of R/M-HNSCC patients. In addition to chemotherapy, cetuximab combined with the CDK4/6 inhibitor palbociclib, 423 VEGF monoclonal antibody bevacizumab, 424 or immunotherapy (pembrolizumab 425 and nivolumab 426 ) also showed promising clinical activity and safety profiles in R/M-HNSCC patients, including platinum-resistant and cetuximabresistant patients.
In the phase 3 SPECTRUM clinical study, the addition of panitumumab to cisplatin and fluorouracil resulted in longer median PFS (5.8 months vs. 4.6 months) than chemotherapy alone in R/M-HNSCC patients, especially in patients with p16-negative tumors. 427 Meanwhile, panitumumab and paclitaxel combination as first-line treatment contributed to almost 50% of confirmed ORR and median PFS of 7.5 months in R/M-HNSCC patients, comparable to the previously reported efficacy of cetuximab-paclitaxel or PCF regimes. 26,418,428 Panitumumab, docetaxel, and cisplatin may have the potential to improve PFS by 1.4 months in R/M-HNSCC patients. However, there is a tendency for a decrease in OS in the panitumumab-containing regimen compared to chemotherapy alone. 429 Therefore, further studies are necessary to evaluate the advantages and disadvantages of this triplet combination. A combination of nimotuzumab, cisplatin, and fluorouracil also demonstrated promising efficacy, including both overall response, survival outcomes, and tolerability in recurrent metastatic NPC (R/M-NPC) patients. 430 These results are consistent with a retrospective study evaluating the antitumor activity and toxicity of nimotuzumab in combination with chemotherapy as first-treatment in 203 R/M-NPC patients. 431 In a phase 3 study performed on 486 patients, gefitinib monotherapy failed to improve the ORR and survival outcomes in R/M-HNSCC patients when compared to methotrexate. 432 The addition of gefitinib to docetaxel also failed to improve the clinical outcomes of R/M-HNSCC patients with poor prognosis. 433 Thus, gefitinib may be used with caution in R/M-HNSCC patients. Erlotinib monotherapy is well tolerated and yields prolonged disease stabilization in heavily pretreated R/M-HNSCC patients. 434 Erlotinib in combination with the chemotherapeutic cisplatin with 435 or without 436 docetaxel exhibited favorable antitumor activity and tolerability comparable to historical controls in R/M-HNSCC, supporting further evaluation of these regimens. Dualtarget inhibitors, such as afatinib and dacomitinib, are also under clinical evaluation against R/M-HNSCC. As an irreversible blocker of the ErbB family, afatinib monotherapy induced significantly prolonged PFS (2.6 vs. 1.7 months) versus methotrexate as second-line treatment in R/M-HNSCC patients with manageable safety profiles in the phase 3 LUX-Head & Neck 1 study. 437,438 The most common serious adverse effects were afatinib-related skin rash/acne and diarrhea. Subgroup analysis indicated that median PFS favored afatinib in patients with p16-negative, EGFRamplified, HER3-low, and PTEN-high tumors. However, in patients with p16-positive disease, afatinib failed to display an advantage in enhancing PFS. 439 Therefore, it is necessary to examine the p16 status of R/M-HNSCC before giving afatinib treatment to guarantee patients' benefits. Similar results were obtained from the LUX-Head & Neck 3 study carried out in an Asian population, demonstrating the superiority and feasibility of afatinib over methotrexate as a second-line treatment for R/M-HNSCC patients. 440 Afatinib may also improve the efficacy of pembrolizumab by promoting antigen presentation machinery in the tumor microenvironment. 441 Dacomitinib is another paninhibitor of the ErbB family that demonstrated clinical activity and acceptable toxicity in platinum-refractory HNSCC patients. 442,443 Poziotinib, a dual inhibitor of EGFR and HER2, exhibited clinically meaningful efficacy (ORR: 24%, median PFS: 4.0 months, median OS: 7.6 months) in R/M-HNSCC patients, 444 which was noninferior to that induced by afatinib monotherapy. 437,438 VEGF inhibitors Sorafenib is an orally active inhibitor of multiple kinases, including BRAF, VEGFR1, and VEGFR-2, which can inhibit cancer cell proliferation and angiogenesis. 445,446 In the phase 3 DECISION study, sorafenib significantly improved the ORR (12.2% vs. 0.5%) and PFS (10.8 vs. 5.8 months) versus placebo in patients with locally advanced or metastatic, radioactive iodine-refractory, differentiated thyroid cancer. 447 Most AEs were grade 1 or 2, and no unexpected AEs occurred. Based on the promising results, sorafenib received FDA and EMA approval for the treatment of radioactive iodine-refractory or metastatic differentiated thyroid cancer (DTC). Further analysis of the DECISION study revealed that elevated baseline levels of VEGFR and thyroglobulin and the presence of RAS mutations were associated with poor PFS, whereas BRAF mutations were related to better PFS. 448 In R/M-NPC patients, sorafenib showed only modest anticancer activity, with an ORR of 3.7% and an OS of 4.2 months. 449 To improve the efficacy, sorafenib was combined with cisplatin and 5-fluorouracil. This triplet combination strategy is tolerable and highly effective in treating R/M-NPC patients, with the ORR reaching 77.8% and favorable survival outcomes (PFS: 7.2 months, OS: 11.8 months). 450 Lenvatinib is an oral inhibitor of VEGFR1/2/3, FGFR1, plateletderived growth factor receptor α (PDGFRα), RET, and c-kit. In the phase 3 SELECT study, lenvatinib yielded a significantly extended PFS (18.3 vs. 3.6 months) and improved response rate (64.8% vs. 1.5%) compared with placebo, including elevated complete response (1.5% vs. 0%), in radioiodine-refractory thyroid cancer patients of any age. 451,452 This study promoted the approval of lenvatinib in treating patients with progressive DTC that progressed after radioactive iodine therapy. Despite the promising results from DTC, lenvatinib seems to be disappointing in treating anaplastic thyroid cancer. 453,454 Sunitinib and apatinib are also multitarget inhibitors of VEGFR, PDGFR, and c-kit, which have been approved for the management of gastrointestinal stromal tumors, renal cell carcinoma, pancreatic cancer, and lung cancer. [455][456][457][458][459] Sunitinib monotherapy only showed modest clinical activity in R/M-HNSCC patients and was associated with a high incidence of hemorrhage. 460,461 Although sunitinib may be effective in managing thyroid cancer, serious side effects have also been reported, including asthenia/fatigue and mucosal and cutaneous toxicities. 462,463 Further studies should focus on different combination strategies to improve sunitinib's efficacy and safety. In the REALITY placebo-controlled phase 3 clinical trial, apatinib monotherapy exhibited significant promising benefits in both enhanced ORR (54.3% vs. 2.2%) and prolonged survival (both PFS and OS) with favorable safety profiles, strongly supporting the application of apatinib in LA-or metastatic radioactive iodine-refractory DTC patients. 464 Apatinib may also show efficacy in patients with R/M-NPC [465][466][467] and locally advanced oral squamous cell carcinoma 468 alone or in combination. Other multitarge inhibitors, including anlotinib 469-471 and donafenib, 472 have shown antitumor activity with favorable safety in thyroid cancer patients in early phase clinical trials. More studies are warranted to confirm these results.
Bevacizumab is a humanized anti-VEGF monoclonal antibody approved for the treatment of various cancer types, including colon cancer, lung cancer, and breast cancer. Although bevacizumab has not been licensed for use in HNC, preclinical and clinical studies have confirmed its effectiveness and tolerability in improving the outcomes of HNC patients. The addition of bevacizumab to platinum-based chemotherapy 473 or nonplatinum pemetrexed 474 significantly improved the response rate (30)(31)(32)(33)(34)(35).5%) and survival (OS: 11.3-12.6 months) compared to chemotherapy alone in R/M-HNSCC patients. However, it should be noted that bevacizumab treatment may result in a significantly increased rate of serious bleeding. Whether bevacizumab can increase the sensitivity of HNSCC patients to chemoradiotherapy remains controversial. It has been reported that the addition of bevacizumab to standard chemoradiotherapy (radiotherapy + cisplatin + fluorouracil) is effective and highly tolerable in LA-NPC patients with no hemorrhage of grade 3 to 5. The 2-year OS and PFS reached 90.9% and 83.7, respectively, with a median follow-up of 2.5 years. 475 However, a phase 2 clinical trial evaluating the effect of bevacizumab plus a different chemoradiotherapy regimen (5-fluorouracil, hydroxyurea, and radiotherapy) in intermediate-stage and T4N0 HNC was terminated in advance because of locoregional progression in the bevacizumab arm, 476 suggesting the potential of bevacizumab to promote tumor progression in this regime. Accumulating studies have also indicated that there is crosstalk between the EGFR and VEGF pathways and upregulation of VEGF-related angiogenesismediated resistance to EGFR inhibition. 477 Consistently, simultaneous inhibition of EGFR (cetuximab or erlotinib) and VEGF pathways (bevacizumab) was reported to be well tolerated and active in R/M-HNSCC patients, with an ORR of 15-16%, PFS of 2.8-4.1 months and OS of 7.1-7.5 months, indicating bevacizumab's role in improving patients' sensitivity to EGFR-targeted therapy. 424,478 Although dual EGFR and VEGF inhibition combined with concurrent chemoradiation is effective in LA-HNSCC patients, 479 a head-to-head phase 2 study indicated that the addition of bevacizumab failed to bring additional efficacy to cetuximab-containing chemoradiotherapy. 480 In contrast, the addition of bevacizumab increased the hemorrhagic rate. 480 Therefore, more studies concerning the effect of bevacizumab on chemoradiotherapy in HNSCC patients are warranted, either alone or in combination with EGFR-targeted agents.
Therefore, the VEGFR pathway is a promising therapeutic target in HNSCC, especially in thyroid cancer. Further studies should focus on minimizing unwanted adverse effects, especially bleeding events, by developing more tolerable agents with biomarkerdriven studies.
Inhibition of the PI3K/AKT/mTOR pathway PI3K inhibitors. PI3K inhibitors can be divided into two different types: ATP-competitive (e.g., alpelisib, buparlisib, copanlisib) and noncompetitive (PX-866) inhibitors. Almost all the inhibitors are panclass I inhibitors except for alpelisib, which is isoform-specific and targets only the p110α of PI3K. 37 Clinical evaluation of PI3K inhibitors in HNC is mainly in early phase clinical trials. In R/M-HNSCC, the addition of buparlisib to cetuximab is well tolerated and significantly improved the disease control rate (49% vs. 91%) and prolonged survival outcomes (PFS: 63 vs. 111 days; OS: 143 vs. 206 days) compared to buparlisib monotherapy. 481,482 Similarly, a combination of buparlisib and paclitaxel showed manageable safety profiles and superiority in median PFS (3.5 vs. 4.6 months) in R/M-HNSCC patients who received previous platinum treatment, indicating the potential of this combination as a second-line treatment strategy. 483 Biomarker analysis of the molecular alterations revealed that TP53 alterations, HPV-negative status, low mutational load, or high infiltration of CD8 T cells are indicators of survival benefit after treatment with buparlisib and paclitaxel. 484 Based on these promising results, a randomized phase 3 clinical trial is recruiting 483 patients to assess the efficacy and safety of buparlisib plus paclitaxel compared to paclitaxel alone in R/M-HNSCC patients (NCT04338399). Two phase 1 studies indicated that alpelisib is effective and has a manageable safety profile at dosages of 200 mg-250 mg when combined with cisplatin-based chemoradiotherapy or cetuximab plus radiotherapy in LA-HNSCC patients. 485,486 Further clinical trials are needed to evaluate its efficacy in HNC patients. However, according to the currently available data, copanlisib and PX-866 demonstrated unfavorable toxicity or no improvement in clinical outcomes when combined with cetuximab in R/M-HNSCC patients, regardless of HPV infection status. 487,488 AKT inhibitors. Only three AKT inhibitors have been investigated against HNC, including MK-2206, perifosine, and ipatasertib. Serious adverse effects of AKT inhibitors include skin rash, hyperglycemia, fatigue, and ulcerative keratitis. [489][490][491] Clinical data are still insufficient about the efficacy of AKT inhibitors in HNC. In a phase 2 clinical trial, perifosine failed to show single-agent activity in R/M-HNSCC patients, hindering further exploration of perifosine as monotherapy in this disease. 492 The results of two ongoing phase 2 clinical trials will help us better understand how AKT inhibitors act in HNC patients (NCT01306045 and NCT05172258). mTOR inhibitors. Current research on mTOR inhibitors in HNC has concentrated on two analogs of rapamycin, everolimus, and temsirolimus. In HNSCC patients, whether everolimus treatment brings more benefits than drawbacks remains controversial. Some phase 2 clinical trials indicated that everolimus had limited activity with a low response rate as a monotherapy or in combination with erlotinib. [493][494][495] Two studies investigating the efficacy and safety of everolimus in combination with cisplatin and radiotherapy were even terminated as a result of funding problems and toxicity (NCT01057277, NCT01009346). However, other phase 2 studies reported that everolimus treatment displayed significant antitumor effects in aggressive radioiodine-refractory or advanced follicular-derived thyroid cancer, with a high rate of disease control, relatively low toxicity profile, and median PFS of 9 months. 496,497 For temsirolimus, in the TEMHEAD phase 2 study, temsirolimus yielded modest antitumor activity in R/M-HNSCC patients with a PFS of 56 days and OS of 152 days, 498 which is similar to the history of single-agent cetuximab in platinum-failed patients. 414 Baseline caspase 3 activity is inversely correlated with PFS in temsirolimus-treated HNSCC patients, making it a potentially useful noninvasive biomarker of sensitivity to mTOR inhibitors. 499 Recently, studies have focused on exploring effective combination strategies for temsirolimus. In a phase 2 study, temsirolimus plus low-dose chemotherapy (carboplatin and paclitaxel) induced a relatively high response rate (41.7%) and long survival (OS: 12.8 months; PFS: 5.9 months) compared to other treatments in R/M-HNSCC patients. 500 However, there were no patients receiving only chemotherapy in this study, making it difficult to evaluate the additional benefits of temsirolimus. In another randomized phase 2 study, a combination of temsirolimus and cetuximab induced a significantly high response in cetuximab-resistant R/M-HNSCC patients compared with temsirolimus alone, although no significant improvement in PFS was observed, indicating that dual blockade of mTOR and EGFR pathways may be a potential strategy to overcome EGFR resistance. 501 C-MET inhibitors C-MET overexpression is common in HNSCC patients, and overactivation of MET signaling contributes to resistance to anti-EGFR therapy. 108,502 Most c-MET inhibitors are multitarget inhibitors that mainly inhibit the function of c-MET. For instance, cabozantinib is an oral inhibitor of multiple kinases, including c-MET, VEGFR-2, RET, KIT, AXL, and FLT3. 503 In an international, randomized, placebo-controlled phase 3 clinical trial, cabozantinib monotherapy (140 mg/d) achieved a significant increase in objective response rate and PFS with acceptable toxicity in 330 patients with metastatic MTC. 504,505 Prolonged PFS can be observed in all subgroups of patients with different ages, previous treatments and RET mutation statuses. 504,506 Based on these results, in November 2012, cabozantinib received FDA approval for the treatment of metastatic MTC. Cabozantinib as salvage therapy for radioiodine-refractory DTC who progressed after treatment with VEGFR targeted therapy also increased the objective response rate by 15% and prolonged PFS, indicating the potential of cabozantinib as a treatment option for thyroid cancer patients with no available standard of care. 507,508 Therefore, cabozantinib was soon approved as a second-line treatment for adult and pediatric patients with LA-or metastatic DTC who are ineligible or refractory to radioactive iodine. In contrast, other c-MET inhibitors, including tivantinib and foretinib, failed to show clinical benefits over risk in patients with R/M-HNSCC alone or combined with cetuximab. 509,510 RET inhibitors Selpercatinib (LOXO-292) is an ATP-competitive, highly selective small molecule inhibitor of RET kinase. In the phase 1/2 LIBRETTO-001 trial, selpercatinib exhibited durable efficacy with mostly lowgrade adverse effects in patients with RET-altered thyroid cancer, including RET-mutant medullary thyroid cancer and RET fusionpositive thyroid cancer. 511 The response rate and PFS were drastically high in all groups of patients, regardless of previous vandetanib or cabozantinib treatment. Owing to the promising results, selpercatinib received accelerated approval for the treatment of patients with RET-mutant MTC or advanced or metastatic RET fusion-positive thyroid cancer in 2020. Since its initial approval, selpercatinib has altered the treatment paradigm for cancer patients with RET mutations. In 2022, the approval of selpercatinib has been expanded to all kinds of solid tumors with a RET gene fusion. Full approval of selpercatinib may be contingent on the confirmation of the clinical benefits from several ongoing phase 3 clinical trials (NCT04819100, NCT04211337, NCT04194944). Vandetanib is a once-daily oral triple-inhibitor of the RET, VEGFR, and EGFR signaling pathways. Vandetanib monotherapy successfully increased the objective response and survival of patients with advanced or metastatic thyroid cancer, with the estimated PFS reaching up to 30.5 months compared to placebo (19.3 months). 512 Soon, the favorable efficacy of vandetanib promoted its FDA approval in treating advanced MTC in 2011. Pralsetinib is another selective and strong inhibitor of RET that also showed clinical activity and safety in RETaltered thyroid cancers in a phase 1/2 study. 513 Further studies are needed to confirm its performance in the clinic.
Other targeted therapies Some other signaling pathways can be targeted and evaluated in clinical trials for HNC, such as activin receptor-like kinase 1 (ALK1), CDK, STAT3, and indoleamine 2,3-dioxygenase 1 (IDO1). A comprehensive understanding and inhibitor directions for targeting signaling pathways in clinical trials in HNC treatment are shown in Fig. 6. Most of these therapeutic agents are still under clinical trials with limited data released about their efficacy and safety. Epacadostat is a strong and selective inhibitor of the IDO1 enzyme. It showed favorable safety and antitumor efficacy in combination with pembrolizumab in multiple advanced solid tumors, including HNSCC, in a multicenter, open-label phase 1/2 trial. 514 A phase 3 study is ongoing to assess the efficacy and safety of epacadostat plus pembrolizumab, pembrolizumab monotherapy, and the EXTREME regimen in R/M-HNSCC patients (NCT03358472), the results of which will help us gain a comprehensive understanding of the clinical activity of epacadostat. Palbociclib, an inhibitor of CDK4/6, showed synergistic effects with cetuximab in patients with platinum and cetuximab doubleresistant, HPV-unrelated HNSCC in a phase 2 study, suggesting its role in overcoming resistance to EGFR-targeted therapy. 423 Dalantercept is a blocker of ALK1 signaling and angiogenesis, which showed a favorable safety profile but only modest activity in R/M-HNSCC patients as monotherapy. 515

CONCLUSION
Although aggressive treatment regimens of inhibitors, chemotherapy, radiotherapy, and surgical resection have been used in HNC, the treatment response in HNC is poor without significant improvement. The heterogeneity status of HNC led to the development of novel and specific effectors for identifying superior therapeutic regimens. Over the past decades, a comprehensive understanding of the pathogenesis, drug resistance mechanism, and alterations have been investigated in HNC. Various novel small molecular inhibitor monotherapies or combinations with other regimens have been developed in HNC in preclinical models and clinical trials. These new drugs have demonstrated remarkable responses and moderate adverse effects by inhibiting targeted gene expression. Compared with traditional regimens such as chemotherapy and radiotherapy, small molecular inhibitors result in fewer adverse effects and improve the tolerance of patients.
In this review, classic targets, such as the PI3K/AKT/mTOR pathway, EGFR signaling, VEGF signaling, FGFR signaling, and MEK/ERK signaling, are discussed. Some novel targets, including MET signaling, CDK4/6 signaling, and Notch signaling, were also involved in this research and need further investigation. EGFR signaling in HNC is the central signaling pathway in targeted treatment. EGFR is an essential effector in convergent signaling pathways, which is also the basis of oncogenic signaling inhibition. TP53 is one of the prominently mutated genes in HNC and is closely related to tumor progression, recurrence and therapeutic resistance. TP53 plays a critical role in regulating HNC. Other gene alterations facilitating the process of HNC are also discussed in this review.
Radiotherapy plays an important role in treating HNSCC. Furthermore, radioresistance leads to the treatment failure of advanced-stage HNSCC. Inhibitors promoting radiosensitivity are worth thoroughly research. Inhibitors of the PI3K/AKT/mTOR pathway, EGFR signaling, VEGF signaling, FGFR signaling, MEK/ ERK signaling, MET signaling, CDK4/6 signaling, and Notch signaling have been investigated in promoting radiosensitivity. Chemotherapy and chemoradiotherapy have been shown to improve clinical outcomes and are considered standard-of-care strategies for HNSCC. Cisplatin-based regimens were the most commonly used combination as a conventional treatment strategy or ICT. However, chemotherapy-induced toxicity is a major concern in HNSCC treatment. Therefore, efforts are focusing on developing multicombination therapy with improved efficacy and safety. Great success has been achieved with the development of cetuximab, which has been approved for the treatment of Fig. 6 Comprehensive understanding and inhibitor direction for targeting signaling pathways in clinical trials in HNC treatment. EGFR epidermal growth factor receptor, MET mesenchymal-epithelial transition factor, AKT serine/threonine-specific protein kinase, mTOR mammalian target of rapamycin, CDK cyclin-dependent kinase, VEGF vascular endothelial growth factor, JAK jenus-activated kinase, STAT signal transducer and activator of transcription, mAb monoclonal antibody, RET rearranged during transfection, p phosphorylation R/M-HNSCC patients in combination with platinum-based therapy and fluorouracil as first-line therapy. Inspired by the triad, scientists are exploring new combination strategies involving other targeted therapeutics and chemotherapeutics. Some results are promising, while others are obscure. For instance, a combination of panitumumab, cisplatin, and fluorouracil prolonged survival in R/M-HNSCC patients when compared with chemoradiotherapy. 427 In contrast, it has been reported that adding cetuximab to the TPF ICT regimen showed no superiority in clinical outcomes over ICT alone in the DeLOS-II trial. 397 Therefore, more studies are necessary to guide the application of targeted therapies and chemotherapies. CDK4/6 inhibition provides new hope for HPV-negative tumor patients. The response to CDK4/6 inhibitors in HPV-negative HNSCC patients was favorable. 516 HPV-positive HNSCC patients could not benefit from CDK4/6 inhibitors. However, it seems that not all HPV-negative HNSCC patients could benefit from inhibitors. Further investigation of CDK4/6 inhibitors in HPV-negative HNSCC patients should be developed. 517,518 The mechanism of CDK4/6 resistance was also explored, which provided considerate predictive biomarkers in predicting the prognosis of the CDK4/6 response.
RET inhibitors have shown remarkable effects in clinical trials. Owing to the promising results, selpercatinib received accelerated approval for the treatment of patients with RET-mutant MTC or advanced or metastatic RET fusion-positive thyroid cancer in 2020. In 2022, the approval of selpercatinib has been expanded to all kinds of solid tumors with a RET gene fusion. Further administration of RET inhibitors is expected.
Although targeted therapies have achieved satisfactory effects in selected patients with specific alterations, the effect of some targeted therapies is far from desirable. Many inhibitors have been approved for treating patients with EGFR alterations. Gefitinib is reported to be effective in NSCLC patients with EGFR exon 19 deletions or exon 21 (L858R) mutations, which might result in undesirable effects in~15% of HNSCC patients. 519 In patients with an unfavorable response rate to EGFR-targeted therapies, some articles demonstrated that these patients may also develop drug resistance. 520,521 It was reported that multiple downstream effectors served as substitutive signaling and were discovered to be continuously activated, which led to EGFR inhibitor resistance. 522 Only 5% of HNC patients have EGFR alterations, which may contribute to the limited effectiveness of tyrosine kinase inhibitors. 523 Genetic sequencing was used to identify specific targets among various types of cancers. However, enormous genetic heterogeneity and precise alterations in genomes result in targeted therapy failure in specific selected humans. Therefore, it is important to identify the heterogeneity of tumors to help choose efficient small molecule inhibitors.
The response to targeted therapies relies on its specific target in tumor tissue. However, off-target side effects may lead to treatment failure and severe adverse effects. 524 The bystander effect may also result in treatment failure. Antibody-directed enzyme prodrug therapy consists of a tumor-specific antibody and a drug-activating enzyme, which may promote the antibody action on targeted tissues. [525][526][527] Some small molecule inhibitors belonging to antibody-directed enzyme prodrug therapy exist between antigen-positive and antigen-negative cancer cells, leading to the bystander effect. 525 The bystander effect could be avoided by regulating the interval time between the enzyme and drugs.
This information offered new insights into treating HNC and different cancers at distinct sites with various pathological subtypes that can be treated by various regimens. The rate of survival and severe toxicities was improved after novel treatment regimens. Multiple studies in progress could contribute to the specific use of novel compounds in therapeutic strategies and precisely identify patients with favorable outcomes via relatively predictive effectors. Due to moderate adverse effects, small molecular inhibitors are still in urgent demand, but the treatment response needs further improvement. Collectively, thorough and comprehensive studies offer more knowledge about the mechanisms of HNC, which has a potential role in preventing and treating HNC. The clinical translation of new inhibitors is still crucial, and combining new agents with traditional regimens also has great potency in treating HNC.